Bactericidal/permeability-increasing protein (BPI) is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. amino acid sequence is set out in SEQ ID NO: 2 hereto. U.S. Pat. No. 5,198,541 and WO89/10486 (PCT/US88/02700) disclose recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI.
BPI is a member of a gene/protein family of lipopolysaccharide (LPS) binding and lipid transfer proteins whose other currently known members include lipopolysaccharide binding protein (LBP), cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP).
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD possesses essentially all the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).]
The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on lipopolysaccharide (LPS). In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. Bacterial LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.
A variety of BPI protein products as described herein have been discovered and produced, including naturally and recombinantly produced BPI holoprotein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides.
BPI protein products are also capable of neutralizing the endotoxic properties of bacteria and their LPS to which these products bind. Because of the gram-negative bactericidal properties and the ability to bind to, clear and neutralize bacterial LPS, BPI protein products can be utilized for the treatment of mammals suffering from diseases caused by gram-negative bacteria, including sepsis, bacteremia, and bacterial endotoxemia. In addition BPI was discovered to have killing and/or inhibitory activities alone, or in combination with other agents, against gram-positive bacteria, mycobacteria, chlamydia, protozoans and fungi. These multiple anti-infective properties make BPI protein products particularly useful and advantageous for anti-infective therapeutic administration.
One BPI amino-terminal fragment, comprising approximately the first 199 amino acid residues of the human BPI holoprotein and referred to as "rBPI.sub.23 " (see Gazzano-Santoro et al., 1992, Infect. Immun. 60: 4754-4761) has been produced by recombinant means as an approximately 23 kD protein. rBPI.sub.23 retains the antibacterial activity against gram-negative organisms and also the LPS-binding/neutralizing activity of BPI. A modified N-terminal fragment, engineered for increased stability and homogeneity has been designated rBPI.sub.21 .DELTA.cys or rBPI.sub.21, and is the expression product of DNA encoding from about amino acid 1 to about 193 or 199 of the N-terminal amino acids of BPI holoprotein, but wherein the cysteine at residue number 132 is substituted with alanine.
Three separate functional domains within the N-terminal region of BPI have been discovered [see, e.g., WO94/20532 (PCT/US94/02465); WO95/19372 (PCT/US94/10427); Little et al., J. Biol. Chem. 269:1865 (1994), hereby incorporated by reference]. These BPI functional domains comprise specified subregions of the amino acid sequence of BPI that contribute to the total biological activity of the protein. Proteolytic cleavage fragments, overlapping 15-mer peptides and other synthetic peptides have been prepared and analyzed. Domain I is defined as comprising the amino acid sequence of BPI from about amino acid 17 to about amino acid 45. Domain II is defined as comprising the amino acid sequence from about amino acid 65 to about amino acid 99. Domain III is defined as comprising the amino acid sequence of BPI from about amino acid 142 to about amino acid 169. The biological activities of functional domain BPI-derived peptides may include LPS-binding, LPS-neutralization, heparin binding, heparin neutralization or antimicrobial activity, including antibacterial and antifungal activities. These peptides, particularly Domain III-derived peptides possess antifungal activity [see also, e.g., WO96/08509 (PCT/US95/09622) and WO 97/04008 (PCT/US96/03845).
Several BPI protein products (i.e., rBPI.sub.23, rBPI.sub.21 which is a BPI analog protein) have been introduced into human clinical trials. Proinflammatory responses to injected endotoxin were significantly ameliorated when rBPI.sub.23 was administered to human volunteers. Thus, humans with endotoxin in circulation may be effectively treated with BPI protein products as described in U.S. patent application Ser. No. 08/291,112 and WO95/19784 (PCT/US95/01151). rBPI.sub.2, is currently in multiple clinical trials for the treatment of severe pediatric meningococcemia, infections complications of hemorrhage due to trauma, infectious complications of liver surgery, severe intra-abdominal infections and antibiotic resistant infections in cystic fibrosis.
A number of other important biological activities of BPI protein products have been discovered. For example, BPI protein products have been shown to have heparin binding and heparin neutralization activities in WO94/20128 (PCT/US94/02401), U.S. Pat. Nos. 5,348,942 and 5,639,727, and U.S. patent application Ser. No. 08/466,624, the disclosures of which are incorporated by reference herein. These heparin binding and neutralization activities of BPI protein products are significant due to the importance of current clinical uses of heparin. Heparin is commonly administered in doses of up to 400 U/kg during surgical procedures such as cardiopulmonary bypass, cardiac catherization and hemodialysis procedures in order to prevent blood coagulation during such procedures. When heparin is administered for anticoagulant effects during surgery, it is an important aspect of post-surgical therapy that the effects of heparin are promptly neutralized so that normal coagulation function can be restored. Currently, protamine is used to neutralize heparin. Protamines are a class of simple, arginine-rich, strongly basic, low molecular weight proteins. Administered alone, protamines (usually in the form of protamine sulfate) have anti-coagulant effects. When administered in the presence of heparin, a stable complex is formed and the anticoagulant activity of both drugs is lost. However, significant hypotensive and anaphylactoid effects of protamine have limited its clinical utility. Thus, due to its heparin binding and neutralization activities, BPI protein products have utility as a substitute for protamine in heparin neutralization in a clinical context without the deleterious side-effects which have limited the usefulness of the protamines (see, e.g., WO94/20128 (PCT/US94/02401) and U.S. Pat. No. 5,348,942). rBPI.sub.23 has been shown to neutralize the anticoagulant effects of administered heparin in human volunteers. The additional anti-infective properties, including antibacterial and anti-endotoxin effects, of BPI protein products are also useful and advantageous in post-surgical heparin neutralization compared with protamine.
Additionally, BPI protein products are useful in inhibiting angiogenesis due in part to their heparin binding and neutralization activities (see, e.g., WO94/20128 (PCT/US94/02401) and U.S. patent application Ser. No. 08/466,624). In adults, angiogenic growth factors are released as a result of vascular trauma (wound healing), immune stimuli (autoimmune disease), inflammatory mediators (prostaglandins) or from tumor cells. These factors induce proliferation of endothelial cells (which is necessary for angiogenesis) via a heparin-dependent receptor binding mechanism (see Yayon et al., 1991, Cell 64: 841-848). Angiogenesis is also associated with a number of other pathological conditions, including the growth, proliferation, and metastasis of various tumors; diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-immune inflammation including rheumatoid arthritis, capillary proliferation within atherosclerotic plaques, hemangiomas, endometriosis and Kaposi's sarcoma. Thus, it would be desirable to inhibit angiogenesis in these and other instances, and the heparin binding and neutralization activities of BPI are useful to that end.
Another utility of BPI protein products involve pathological conditions associated with chronic inflammatory disease states, which are usually accompanied by angiogenesis (see, e.g., WO94/20128 (PCT/US94/02401) and U.S. Pat. No. 5,639,727). One example of a human chronic inflammatory disease state is arthritis, which involves inflammation of peripheral joints. In rheumatoid arthritis, the inflammation is immune-driven, while in reactive arthritis, inflammation is associated with infection of the synovial tissue with pyogenic bacteria or other infectious agents. Folkman et al., 1992, supra, have also noted that many types of arthritis progress from a stage dominated by an inflammatory infiltrate in the joint to a later stage in which a neovascular pannus invades the joint and begins to destroy cartilage. While it is unclear whether angiogenesis in arthritis is a causative component of the disease or an epiphenomenon, there is significant evidence that angiogenesis is necessary for the maintenance of synovitis in rheumatoid arthritis. While nonsteroidal anti-inflammatory drugs, corticosteroids and other therapies have provided treatment improvements for relief of arthritis, there remains a need for more effective therapies, such as treatment with BPI protein products, for arthritis and other chronic inflammatory disease states.
BPI is also known to possess biological activity useful for the treatment of thrombotic disorders. BPI protein products reduce the adverse effects of thrombotic disorders by activites that include slowing or delaying clot formation (i.e., anticoagulant activity) and/or by enhancing, accelerating or increasing clot dissolution (i.e., thrombolytic activity). Thus, BPI protein products are useful in methods for the treatment of thrombotic disorders, for dissolving or lysing clots in thrombotic patients, for delaying or inhibiting hard clot formation or supplementing thrombolytic therapy in the patients (see, e.g., U.S. patent application Ser. No. 08/644,290 and PCT/US97/08017, hereby incorporated by reference).
A need continues to exist for new products and methods for use as anti-infective products, including antimicrobial agents (e.g., gram-negative bacteria [U.S. Pat. Nos. 5,198,541 and 5,523,288; WO95/08344 (PCT/US94/11225)] and gram-positive bacteria [U.S. Pat. No. 5,578,572; WO95/19180 (PCT/US95/00656)], fungi [U.S. Pat. No. 5,627,153; WO95/19179 (PCT/US95/00498)], mycobacteria [WO94/20129 (PCT/US94/02463)] and chlamydia [WO96/01647 (PCT/US95/08624)] and endotoxin binding/neutralizing agents [WO95/019784 (PCT/US95/01151)], and as heparin binding/neutralizing products [U.S. Pat. Nos. 5,348,942 and 5,639,727; WO94/20128 (PCT/US94/02401], including for the neutralization of exogeneously administered heparin, inhibition of angiogenesis (normal or pathological) for the treatment of chronic inflammatory disease states, and anticoagulant and thrombolytic agents for the treatment of thrombotic disorders [PCT/US97/08017]. All of the above-listed references regarding biological activities of BPI are hereby incorporated by reference. One avenue of investigation towards fulfilling this need is the determination of the crystal structure of BPI. Advantageous therapeutic embodiments would therefore comprise therapeutic and/or diagnostic agents based on or derived from the three-dimensional crystal structure of BPI that have one or more than one of the functional activities of BPI. Additional therapeutic embodiments would comprise therapeutic and/or diagnostic agents based on or derived from molecular modeling of other members of the BPI protein family, such as LBP, CETP and PLTP, using three-dimensional crystal structure of BPI.